This application is a continuation on our studies of normal and abnormal hemoglobin synthesis in sickle cell anemia and thalassemia. The project in the previous grant period consisted of three aims. 1) DNA diagnosis of sickle cell anemia and thalassemia. 2) Control of globin gene expression. 3) Globin gene transfer. Because the topics in Aim 2 will now be carried on by previous trainees who have become independent investigators, this proposal will continue to pursue only the first and third aims. hi Aim 1 we have completed our studies on dot blot hybridization for the diagnosis of the mutations in hemoglobinopathies and thalassemia that are commonly found in the American population. We propose to develop and refine methods of prenatal diagnosis from fetal cells isolated using maternal blood. Our preliminary studies show that this is a promising approach, but much more work is needed to make this test practical. We will use phage display to isolate single chain antibodies specific for fetal nucleated red cells and human embryonic hemoglobin. We will investigate the use of image analysis and laser capture microdissections to facilitate the retrieval of fetal nucleated red cells. These developments will greatly enhance the usefulness of this procedure. The second aim of this proposal is to explore the possibility of in utero gene delivery alpha-thalassemia is a good model for in utero gene therapy because pathological changes in homozygous alpha-thalassemia usually appear before birth. In our laboratory, we have made mouse models of alpha-thalassemia by knocking out the endogenous mouse alpha-globin genes. By crossing various strains, we have mice that have 3,2,1 or no alpha-globin gene and they mimic the clinical manifestation of human alpha-thalassemia. We will use AAV vectors and lentiviral vectors containing the beta-g1obin LCR controlling the human a-globin gene to inject into fetal mice at the 15th week of gestation. We will follow these mice to observe the expression of the. alpha-globin gene and the rescue the disease phenotype. Such an approach could be useful not only for alpha-thalassemia but also for other genetic disorders such as OTC deficiency where the disease begins in utero.